A role for amyloid precursor-like protein 2 in corneal epithelial wound healing.
OBJECTIVE
The authors previously showed that the mRNA encoding the amyloid precursor-like protein 2 (APLP2) was one of several genes with upregulated expression in healing corneal epithelium. To elucidate the physiological role of APLP2 in corneal epithelial cells, the expression, distribution, and posttranslational modification of APLP2 were investigated.


METHODS
Alternative splicing was assessed by reverse transcription-polymerase chain reaction; translational expression and posttranslational modification were assessed by chondroitinase digestion and Western blotting. The differential distribution of APLP2 in corneal epithelium was examined by in situ hybridization and immunohistochemical analyses.


RESULTS
In rat corneal epithelium, the majority of APLP2 mRNA generated from two alternative splicing sites was found to contain the exon encoding a Kunitz protease inhibitor domain in the first site but to lack the exon encoding a 12-amino acid insert in the second site. The absence of the 12-amino acid insert indicated that APLP2 could be modified by the addition of a chondroitin sulfate (CS) glycosaminoglycan chain. The CS proteoglycan nature of APLP2 was verified by chondroitinase digestion. After wounding, APLP2 mRNA and polypeptides were increased markedly in the basal epithelial cells that were actively migrating. Furthermore, APLP2 was observed in the denuded wound bed immediately adjacent to the leading edge of migratory cells and under the epithelial sheet after wound closure.


CONCLUSIONS
The wound-induced, basal-cell-specific APLP2 expression correlates with epithelial cell migration. The spatial and temporal expression of Kunitz protease inhibitor-containing, CS-modified APLP2 in healing corneal epithelium is consistent with its hypothesized role(s) in mediating reorganization of the extracellular matrix and dynamic cell-matrix adhesion during reepithelialization.
Guo
J
J
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Thinakaran
G
G
Guo
Y
Y
Sisodia
S S
SS
Yu
F X
FX
D017398:Alternative Splicing; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000502:physiology*; D000818:Animals; D007611:Aprotinin / Q000235:genetics; D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D019573:Epithelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D017403:In Situ Hybridization; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014945:Wound Healing / Q000502:physiology
C489697:APLP1 protein, human; D016564:Amyloid beta-Protein Precursor; C498283:Aplp2 protein, rat; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D007611:Aprotinin